Department of Chemistry and Chemical Biology, Harvard University , Cambridge, Massachusetts 02138, United States.
Department of Microbiology and Immunobiology, Harvard Medical School , Boston, Massachusetts 02115, United States.
J Am Chem Soc. 2018 Feb 21;140(7):2442-2445. doi: 10.1021/jacs.7b13551. Epub 2018 Feb 9.
The Gram-positive bacterial cell wall is a large supramolecular structure and its assembly requires coordination of complex biosynthetic pathways. In the step that merges the two major biosynthetic pathways in Staphylococcus aureus cell wall assembly, conserved protein ligases attach wall teichoic acids to peptidoglycan, but the order of biosynthetic events is a longstanding question. Here, we use a chemical approach to define which of the possible peptidoglycan intermediates are substrates for wall-teichoic acid ligases, thereby establishing the order of cell wall assembly. We have developed a strategy to make defined glycan chain-length polymers of either un-cross-linked or cross-linked peptidoglycan, and we find that wall teichoic acid ligases cannot transfer wall teichoic acid precursors to the cross-linked substrates. A 1.9 Å crystal structure of a LytR-CpsA-Psr (LCP) family ligase in complex with a wall teichoic acid precursor defines the location of the peptidoglycan binding site as a long, narrow groove, and suggests that the basis for selectivity is steric exclusion of cross-linked peptidoglycan. Consistent with this hypothesis, we have found that chitin oligomers are good substrates for transfer, showing that LCPs do not discriminate cross-linked from un-cross-linked peptidoglycan substrates by recognizing features of the un-cross-linked stem peptide. We conclude that wall teichoic acids are coupled to un-cross-linked peptidoglycan chains at an early stage of peptidoglycan synthesis and may create marks that define the proper spacing of subsequent cross-links.
革兰氏阳性菌细胞壁是一个大型超分子结构，其组装需要协调复杂的生物合成途径。在金黄色葡萄球菌细胞壁组装过程中，两条主要生物合成途径融合的步骤中，保守的蛋白连接酶将细胞壁磷壁酸连接到肽聚糖上，但生物合成事件的顺序是一个长期存在的问题。在这里，我们使用化学方法来确定哪些可能的肽聚糖中间体是细胞壁磷壁酸连接酶的底物，从而确定细胞壁组装的顺序。我们已经开发出一种策略，可以制造出未交联或交联的肽聚糖的定义糖链长度聚合物，并且我们发现细胞壁磷壁酸连接酶不能将细胞壁磷壁酸前体转移到交联的底物上。LytR-CpsA-Psr（LCP）家族连接酶与细胞壁磷壁酸前体的 1.9 Å 晶体结构定义了肽聚糖结合位点的位置是一个长而窄的凹槽，并表明选择性的基础是交联肽聚糖的空间排斥。与该假设一致，我们发现几丁寡糖是转移的良好底物，表明 LCP 不是通过识别未交联的茎肽的特征来区分交联和未交联的肽聚糖底物。我们得出结论，细胞壁磷壁酸在肽聚糖合成的早期与未交联的肽聚糖链偶联，并且可能形成标记，定义随后交联的适当间隔。
